Related Experiment Videos
Molecular cloning of lsk, a carboxyl-terminal src kinase (csk) related gene, expressed in leukocytes
D W McVicar1, B K Lal, A Lloyd
1Leukocyte Cell Biology Section, Inc./DynCorp, National Cancer Institute, Frederick Cancer Research and Development Center, Maryland 21702-1201.
Abstract:
Regulation of the activity of src-family kinases is thought to occur, in part, through the phosphorylation of conserved carboxyl-terminal tyrosine residues. Although the src-family includes several molecules with tissue or cell-type restricted expression, the only kinase implicated in the regulatory phosphorylation of these enzymes is p50csk. Herein we report the molecular cloning of a tissue specific p50csk-related gene. Like p50csk, the deduced protein sequence of this novel cDNA includes a tyrosine kinase catalytic domain, SH2 and SH3 domains, a short amino terminus, and no autophosphorylation or carboxyl-terminal tyrosine residues. Additionally, neither this novel kinase nor p50csk contain the amino-terminal myristoylation site characteristic of the src-family. However, whereas csk is ubiquitously expressed, mRNA corresponding to this novel gene is expressed in brain, natural killer (NK) cells, and activated T cells but not in a variety of other tissues and cell lines. In agreement with the mRNA expression pattern, antiserum reactive with the predicted carboxyl-terminus of the cDNA recognizes a 57 kDa polypeptide in immunoblots of NK cells and PHA-activated T cells. Because of its limited expression and high homology to p50csk, we named this gene lsk; leukocyte carboxyl-terminal src kinase related gene. Identification of a molecule like lsk suggests the existence of tissue specific src-regulatory pathways that function in activated lymphocytes.
Insights
Researchers discovered a new gene, lsk (leukocyte carboxyl-terminal src kinase related gene), which is specifically expressed in brain, NK cells, and activated T cells. This finding suggests novel tissue-specific regulatory pathways for src kinases in lymphocytes.
Area of Science:
- Molecular Biology
- Immunology
- Cell Signaling
Background:
- Src-family kinases (SFKs) are regulated by phosphorylation of tyrosine residues.
- p50csk is the known kinase involved in SFK regulation.
- SFKs have restricted tissue expression, but regulatory mechanisms are not fully understood.
Purpose of the Study:
- To identify and characterize novel genes involved in the regulation of src-family kinases.
- To investigate tissue-specific regulation of SFKs.
Main Methods:
- Molecular cloning of a novel cDNA.
- Sequence analysis to identify protein domains (tyrosine kinase, SH2, SH3).
- mRNA expression analysis using Northern blotting.
- Protein detection using Western blotting with specific antiserum.
Main Results:
- A novel p50csk-related gene, named lsk, was cloned.
- Lsk encodes a protein with tyrosine kinase, SH2, and SH3 domains, lacking autophosphorylation and myristoylation sites.
- Lsk mRNA is specifically expressed in brain, NK cells, and activated T cells, unlike ubiquitously expressed csk.
- A 57 kDa Lsk protein was detected in NK and activated T cells.
Conclusions:
- Lsk represents a novel, tissue-specific regulator of src-family kinases.
- The discovery of lsk suggests the existence of distinct src-regulatory pathways in lymphocytes.
- Lsk may play a crucial role in the function of activated immune cells.